Measurement of analytes with membrane channel molecules, and bilayer arrays

ABSTRACT

The invention relates to a method for detection of analyte interaction with a channel molecule held in a membrane, comprising the optical detection of a modification in the flux of a signal molecule as it passes through the channel molecule by the action of a membrane potential, wherein the modification in the flux is caused by at least partial blockage of the channel molecule by the analyte. The invention further relates to bilayer arrays, components, methods of manufacture and use.

ACKNOWLEDGEMENT OF GOVERNMENT FUNDING

This invention was made with government funds under Grant No. RO1HG003709 awarded by National Institutes of Health. The US Government hasrights in the invention.

This invention relates to analysis of analytes, such as biologicalmolecules, by channel molecules in a membrane. The invention furtherrelates to a bilayer array, components thereof, and the manufacture anduse of a bilayer array.

INCORPORATION BY REFERENCE

The description refers to various publications, the contents of whichare incorporated herein by reference.

Introduction

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology, for example as described by Clarke et al (2009.“Continuous base identification for single-molecule nanopore DNAsequencing”. Nature Nanotechnology 4 (4): 265-270.doi:10.1038/nnano.2009.12. PMID 19350039), incorporated herein byreference. When a potential is applied across a nanopore, there is adrop in the current flow when an analyte, such as a nucleotide, residestransiently in the barrel for a certain period of time. Nanoporedetection of the nucleotide gives a current blockade of known signatureand duration. The nucleotide content can then be determined by thenumber of blockade events, or the level of blockade as the nucleotideanalyte passes through a single pore. This approach has been applied fordetermining other analytes, such as amino acid polymers, and formeasuring stochastic interactions (See—Howorka et al. Stochasticdetection of monovalent and bivalent protein-ligand interactions.Angewandte Chemie International Edition 43 (7), 842-846 (2004); andCheley et al. A genetically encoded pore for the stochastic detection ofa protein kinase. ChemBioChem 7 (12), 1923-1927 (2006) incorporatedherein by reference.).

The completion of the first human genome sequencing in 2004, has spurredon the development of various new approaches aiming for sequencing ahuman genome in 15 minutes under $1000. Nanopore sequencing, whichoffers advantages of being label free, amplification free, long readinglength, fast speed and low cost, is of particular interests forinvestigations [Venkatesan, 2011 NatNano]. Intensive studies havefocused on lowering DNA translocation speed for resolving single bases.Recent progress showed that a processive enzyme ratchets DNA movementwith single base resolution [Cherf, 2012 NatBiotech] and base identitiescan be sequentially read out (˜28 ms median duration and ˜40 pA maximumlevel separations) for sequencing [Manrao, 2012 NatBiotech]. With thisspeed, a minimum of 10⁶ parallel recordings are still needed to achieve15 minutes human genome sequencing. However, fabricatingelectrodes/amplifiers array in high density is of technical challengesand is limiting both the throughput and cost of nanopore analysistechnologies.

An aim of the present invention is to provide improved analyte analysisand detection using channel molecules, such as nanopores, and improvedbilayer devices and methods for the analyte analysis.

DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention, there is provideda method for detection of analyte interaction with a channel moleculeheld in a membrane, comprising the optical detection of a modificationin the flux of a signal molecule as it passes through the channelmolecule by the action of a membrane potential, wherein the modificationin the flux is caused by at least partial blockage of the channelmolecule by the analyte.

Advantageously, by using optical measurements of flux the presentinvention enables recordal of the flux through many channel molecules inparallel without the need for multiple complex and expensive arrays ofelectrodes. The optical measurements can mirror an electrical readoutfrom multiple membrane channel molecules, but can be separated intomultiple fluorescent traces for each channel. The method of theinvention can replace electrical recording for applications that requirehigh throughput screening such as a nanopore sequencing array.

The method may comprise forming the membrane, wherein the membranecontains the channel molecules. The membrane may be any material capableof supporting a functional channel molecule, such as a nanopore. Themembrane may be a synthetic membrane, for example in embodiments of theinvention wherein the channel molecules comprise solid-state nanopores.The membrane may comprise or consist of a polymer. The membrane maycomprise or consist of block-copolymers, for example as provided inDischer, D. E. & Ahmed, F. Polymersomes. Annu. Rev. Biomed. Eng. 8,323-341 (2006), incorporated herein by reference. The membrane maycomprise or consist of a solid substrate layer, such as SiN. Themembrane may comprise cross-linking lipids such as 23:2 Diyne PC[DC(8,9)PC] (1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine).

The membrane may comprise a bilayer, such as a bilayer of amphipathicmolecules. The membrane may be synthetic. The membrane may beartificial. The bilayer may be artificial, for example non-natural. Thebilayer may not be a cell bilayer. The bilayer may not be a patch clampbilayer of a cell. The skilled person will understand that there aremultiple methods for providing a bilayer. The bilayer may be provided byany artificial means of forming a bilayer. The bilayer may be providedby a droplet hydrogel bilayer (DHB) method, for example as provided inWO2009024775, the contents of which is incorporated herein by reference.The bilayer may be provided by a hydrogel-hydrogel interaction in ahydrophobic medium comprising amphipathic molecules, such as lipids. Thebilayer may be provided by the bilayer array of the invention herein.Alternatively, other bilayer forming methods are available. For example,the bilayer may be provided by any one of the following techniques knownto the skilled person comprising: patch clamping, for example opticalpatch clamping; black lipid membrane (BLM); otherwise known as paintedBLM; Supported lipid bilayers (SLB); and tethered bilayer lipidmembranes (t-BLM). The bilayer may be formed across an aperture inaccordance with WO2008102121, the content of which is incorporatedherein by reference. The bilayer may be formed at droplet to dropletinterfaces in accordance with WO2014064444, the content of which isincorporated herein by reference.

The bilayer may comprise a Cis side and a Trans side. The channelmolecules, such as αHL nanopores, may be placed on the Cis side of thebilayer and spontaneously insert into the bilayer and conduct the signalmolecule, such as Ca²⁺, from the Trans side of the bilayer into the Cisside.

The channel molecules may be provided in the bilayer by adding channelmolecules to the bilayer after the bilayer is formed, or duringformation of the bilayer. For example, the channel molecule may beprovided in an aqueous suspension or solution in a hydrogel or droplethaving a monolayer of amphipathic molecules, wherein the monolayer isbrought into contact with an opposing monolayer to spontaneously form abilayer, wherein the channel molecules may insert into the bilayer.Channel molecules may be provided to the bilayer by channelmolecule-containing liposomes fusing with the bilayer.

A channel molecule may comprise a transmembrane pore. The channelmolecules may comprise or consist of a nanopore. The channel moleculemay be natural, for example derived from a biological organism, or thechannel molecule may be synthetic. The channel molecule may berecombinantly produced. The channel molecule may be isolated from amembrane of a cell. The channel molecule may be a biological molecule.For example a pore-forming protein in a membrane such as a lipidbilayer.

The channel molecule may comprise alpha-hemolysin (α-HL). The channelmolecule may comprise a modified alpha-hemolysin (α-HL), which iscapable of enhanced detection of specific nucleotides in accordance withWO2010004273, the content of which is incorporated herein by reference.Alternatively, the channel molecule may comprise Mycobacterium smegmatisporin A (MspA). The channel molecule may comprise Mycobacteriumsmegmatis porin A (MspA) modified to improve translocation, for exampleby having neutral asparagine residues in place of three negativelycharged aspartic acids. Phi29 polymerase may be used in conjunction withthe channel molecule.

The MspA pore may be advantageous for DNA sequencing because of itsshape and diameter. For example it has been shown to be tenfold morespecific than αHL for identifying bases.

The channel may be a solid-state channel, for example comprisingsynthetic materials such as silicon nitride or graphene. A solid-statechannel is typically a nanometer-sized hole formed in a syntheticmembrane (usually SiNx or SiO2). The pore can be fabricated by focusedion or electron beams, allowing the size of the pore to be tuned. Thechannel may be a hybrid channel comprising a pore-forming protein set insynthetic material.

Multiple channels may be provided in the membrane. The number ofchannels provided in a single membrane may be controlled such that thechannels are on average, distanced apart by 3 μm or more. For example,the channels may be provided at a density/concentration in the membranesuch that single channels may be optically resolved. The maximum densityof channels in the bilayer may be determined by the accuracy to whichthe point spread function corresponding to optical detection of thenanopore can be determined. For fluorescence based optical detectionthis accuracy may be about 1 nanometre, whereby the maximum density maybe determined to be 1 channel molecule per square nanometre. The channelmolecule may be provided at a concentration of at least one channelmolecule per 2 nm×2 nm. The channel molecule may be provided at aconcentration of at least one channel molecule per 5 nm×5 nm. Thechannel molecule may be provided at a concentration of at least onechannel molecule per 10 nm×10 nm. The channel molecule may be providedat a concentration of at least one channel molecule per 50 nm×50 nm. Thechannel molecule may be provided at a concentration of at least onechannel molecule per 100 nm×100 nm.

The concentration may be limited by the Raleigh diffraction limit forresolving individual channel molecule, for example in the absence ofsuper-resolving the channel molecule position. For example channelmolecule concentration may provide a channel molecule approximatelyevery 200 nm×200 nm. The channel molecule may be provided at aconcentration of at least one channel molecule per 300 nm×300 nm. Thechannel molecule may be provided at a concentration of at least onechannel molecule per 400 nm×400 nm. The channel molecule may be providedat a concentration of at least one channel molecule per 500 nm×500 nm.The channel molecule may be provided at a concentration of at least onechannel molecule per 1000 nm×1000 nm.

Advantageously, the present invention allows measurement of a very highdensity of channel molecules compared to conventional electrical methodsfor measuring signals from channel molecules.

The signal molecule may be a first signal-associated molecule providedon one side of the membrane, wherein the first signal-associatedmolecule is capable of flux through the channel molecule by the actionof the membrane potential across the membrane;

-   -   and a second signal-associated molecule is provided on the        opposing side of the membrane relative to the first        signal-associated molecule, wherein the first and/or second        signal associated molecules are arranged to emit an optical        signal when in contact.

The signal molecule may only cause or provide optical detection afterpassing through the channel molecule. The signal molecule may interactwith one or more other signal associated molecules to cause or provideoptical emission for optical detection after passing through the channelmolecule. For example, the signal molecule may interact with the secondsignal-associated molecule to cause or provide optical emission foroptical detection after passing through the channel molecule. Theoptical emission may be provided upon laser excitation, for example at473 nm.

The signal molecule may be any molecule capable of providing anoptically resolvable signal caused by the transit of the signal moleculethrough the channel molecule. For example, the signal molecule may bearranged to provide a change in refractive index of a material relativeto the surroundings; or a change in optical absorption; or a change influorescence emission.

The signal molecule may be membrane-impermeant, such asbilayer-impermeant. The signal molecule may be an ion. The signalmolecule may comprise electrolytes such as Ca²⁺, Na⁺ or, Mg²⁺. Thesignal molecule may comprise Ca²⁺.

The second signal-associated molecule may be membrane-impermeant, suchas bilayer-impermeant. The second signal-associated molecule maycomprise a dye. The second signal-associated molecule may comprise afluorophore dye.

The second signal-associated molecule may comprise a calcium indicator.A calcium indicator is considered to be a molecule, such as a smallmolecule, that can chelate calcium ions. The second signal-associatedmolecule may comprise a calcium-specific aminopolycarboxylic acid. Thesecond signal-associated molecule may comprise BAPTA(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid).

The second signal-associated molecule may comprise any molecule selectedfrom the group comprising fura-2; indo-1; fluo-3; fluo-4; CalciumGreen-1; Fluo-8; and Fluo-4; or combinations thereof. The secondsignal-associated molecule may comprise Fluo-8 or Fluo-4.

Advantageously, the binding of a Ca²⁺ ion to a fluorescent indicatormolecule leads to either an increase in quantum yield of fluorescence oremission/excitation wavelength shift that can be detected optically.

It will be understood by the skilled person that alternative signalmolecules capable of flux through a channel molecule by the action ofmembrane potential may be envisaged.

In one embodiment of the invention, the signal molecule may be aquenching molecule. For example, an optical signal may be blocked duringflux of the quencher, by action of the quencher on a fluorescingmolecule that is capable of being quenched, wherein the blocking of theflux by the analyte causes less quenching signal molecules to passthrough the channel molecule, thereby causing a detectable increase inthe optical signal. Quenching signal molecules may comprise iodine ions,or nitroyl-quenchers.

Analyte interaction with a channel molecule may be analyte flux throughthe channel molecule. Analyte interaction with a channel molecule may beanalyte insertion into the channel molecule. Analyte interaction with achannel molecule may be a blocking of the channel. Analyte interactionmay be a change in conformation of the channel molecule. Analyteinteraction with a channel molecule may be a stochastic blocking of thechannel molecule. Analyte interaction with a channel molecule may be aspecific blocking of the channel molecule, for example by an analytehaving affinity for the channel molecule. The analyte may be a moleculecapable of flux through the channel molecule under the action of amembrane potential. The analyte may be a molecule capable of at leastinsertion into the channel molecule under the action of a membranepotential. The analyte may be provided on only one side of the membrane.

The analyte may comprise or consist of a biological molecule. Theanalyte may comprise or consist of a peptide or nucleic acid. Theanalyte may comprise or consist of an oligomer, such as anoligonucleotide. The analyte may comprise or consist of apolynucleotide. The polynucleotide may comprise or consist of DNA. Thepolynucleotide may comprise or consist of RNA. The polynucleotide may besingle stranded. The polynucleotide may be double stranded, at least inpart.

The analyte may comprise an aptamer-protein complex. For example, aprotein may be targeted by an aptamer specific for that protein, whereinthe aptamer interacts with the channel molecule to provide acharacteristic modification in flux, thereby identifying the protein.

In an embodiment where the nucleic acid is at least partially doublestranded nucleic acid, the action of flux through the channel moleculemay unzip the double stranded nucleic acid to become single stranded.

The method for detection of analyte interaction with a channel moleculeheld in a membrane may be a method for optical screening of apolynucleotide present in one or more samples, wherein the analyte is apolynucleotide. The polynucleotide analyte may comprise a templatepolynucleotide arranged to hybridise with a target polynucleotide in thesample(s). The template polynucleotide may comprise a complementarytarget sequence, which is intended to be complementary relative to atarget polynucleotide sequence potentially in the sample(s). Thecomplementary target sequence may be known/pre-determined. The templatepolynucleotide may further comprise 5′ and 3′ single stranded tagsequences, which flank the complementary target sequence. The tagsequences may not be arranged to hybridise with any polynucleotide inthe sample.

A reduction in the flux of the signal molecule may be indicative of asuccessful hybridisation with the template polynucleotide as thehybridised double stranded polynucleotide at least partially blocks thechannel molecule. Modification of flux may be detected as the hybridisedtemplate and sample polynucleotide is unzipped as it passes through thechannel molecule. The template polynucleotide and sample derived targetpolynucleotide may be hybridized prior to introduction to the membrane,such as the bilayer. The template polynucleotide may comprise a specificnucleotide sequence known to provide a specific signal duringinteraction with the channel molecule. The optically detectedreduction/blockage of flux through the channel molecule may be due tothe presence of a specific nucleotide sequence entering the channel/poreof the channel molecule. The specific nucleotide sequence may be areporter sequence.

Advantageously, with up to ˜300 Hz frame rate and ˜1 pA amplituderesolution, the method of optical screening could resolve fast kineticprocess like miRNA unzipping in nanopores. miRNA, a short (˜22nucleotides) and non-coding RNA fragment, is of significant biologicalimportance but difficult to be quantitatively analyzed by PCR basedmethods. The miRNA, when hybridized with a DNA probe and electricallystretched in a nanopore, can be forced to unzip. The unzipping kinetics,which can be recorded from a single pore in the membrane may reveal themiRNA identity statistically [Wang, 2011 Nature Nano]. The unzippingduration time is widely distributed and may requires a significantamount of events for statistics. The method of the invention which iscapable of imaging channel molecule activities in massive throughput andstreams of single molecule fluorescent traces simultaneously, is idealfor ultra fast recording and screening of miRNA samples.

The method of optical screening may comprise the simultaneous orparallel screening of multiple samples and/or may comprise the use ofmultiple template polynucleotides. The method for optical screening maybe carried out in an array of membranes. For example, the method ofoptical screening may comprise the use of the bilayer array according toinvention herein, wherein the bilayer array comprises an array ofbilayers comprising the channel molecules held in the bilayers. The sametemplate polynucleotide sequence may be provided on two or more, oreach, membrane of the array. Alternatively or additionally, the samesample polynucleotides may be provided on two or more, or each, membraneof the array. A different template polynucleotide sequence may beprovided on two or more, or each, membrane of the array. Differentsample polynucleotides may be provided on two or more, or each,membranes of the array.

The sample may be, for example, a sample of blood, urine, serum, saliva,cells or tissue. The sample may be an environmental sample. The samplemay comprise any medium potentially comprising polynucleotide sequencesof interest.

The target polynucleotide in the sample may comprise DNA, RNA, mRNA, ormiRNA, such as siRNA. The target polynucleotide in the sample may beless than 100 nucleotides. The target polynucleotide in the sample maybe less than 50 nucleotides. The target polynucleotide in the sample maybe less than 30 nucleotides. The target polynucleotide in the sample maybe less than 25 nucleotides. The target polynucleotide in the sample maybe between about 8 nucleotides and about 50 nucleotides, or betweenabout 8 nucleotides and about 30 nucleotides.

The method for detection of analyte interaction with a channel moleculeheld in a membrane may be a method for optical polymer sequencing. Themethod for detection of analyte interaction with a channel molecule heldin a membrane may be a method for optical polynucleotide sequencing. Theoptical sequencing may be parallel, such as massively parallel,sequencing in an array.

The modification in the flux may be caused by blockage of the channelmolecule by the polynucleotide bases as they pass through the channelmolecule. Different bases may be distinguishable by different levels offlux blockage or interference, which can be correlated to individualbases. For example, bases A, C, G and T may occupy a different spacerelative to each other and thereby block the channel molecule by varyingdegrees.

The optical polynucleotide sequencing may not require the use of labels,such as fluorescent labels tagged to the polynucleotide, oroligonucleotides arranged to hybridise to the polynucleotide to besequenced. However method of the invention may be used in channelmolecule mediated polynucleotide sequencing techniques, which requireoptical measurement of fluorescently labelled probes, for example asdescribed in McNally B, Singer A, Yu Z, Sun Y, Weng Z, Meller A (2010).“Optical recognition of converted DNA nucleotides for single moleculeDNA sequencing using nanopore arrays.” Nano Lett. 10 (6):2237-2244,incorporated herein by reference.

The optical polynucleotide sequencing may comprise the use of themethods described by Clarke et al (2009. “Continuous base identificationfor single-molecule nanopore DNA sequencing”. Nature Nanotechnology 4(4): 265-270. doi:10.1038/nnano.2009.12. PMID 19350039), with opticaldetection of the signal molecule flux.

The polynucleotide analyte may be double stranded, wherein thepolynucleotide is unzipped as it passes through the channel molecule.Double stranded polynucleotides may comprise a hairpin loop to linksense and antisense strands. The hairpin loop may be added to a doublestranded polynucleotide prior to sequencing. The optical polynucleotidesequencing may comprise the use of the hairpin loop method for doublestranded polynucleotide sequencing using the channel in accordance withWO2013014451, the content of which is incorporated herein by reference,whereby the method is modified to provide optical detection of fluxthrough the channel in accordance with the invention herein.

The membrane potential may be provided. The membrane potential may beapplied across the membrane such that the signal molecule, for examplethe first signal-associated molecule, is transported through the channelmolecule and interacts with the second signal-associated molecule toemit an optical signal.

The membrane potential may be provide by electrical means, or chemicalmeans. The membrane potential may be ionic membrane potential. Themembrane potential may be a chemical membrane potential, such as anosmotic membrane potential. Electrodes may be applied to providemembrane potential. For example a cathode and anode may be applied toprovide the membrane potential. A cathode may be applied to one side ofthe membrane and an opposing anode may be applied to the opposite sideof the membrane. A single set of electrodes, that is a cathode and ananode, may be applied. The method may not comprise the use of multiplesets of electrodes. In an embodiment comprising an array of membranes, asingle means of providing the membrane potential may apply the membranepotential for all membranes in the array, or groups of membranes in thearray. The array of membranes may be served by a common electrode orshare a common buffer to provide a chemical membrane potential. Forexample in a bilayer array provided by multiple hydrogel pillarsopposing a hydrogel surface, the discrete bilayers may be formedtherebetween, and all hydrogel pillars may be electrically or chemicallyconnected through the hydrogel.

The skilled person will understand that there many methods of setting upa membrane potential across a membrane and such methods may be appliedin the present invention.

The emitted optical signal may be detected. Any modification or lackthereof, in the optical signal may be detected as the flux of the signalmolecule is modified by at least partial blocking of the channelmolecule by the analyte as it interacts with the channel molecule.

Optical detection may comprise microscopy or spectroscopy of themembrane and membrane region. Optical detection may comprise the use ofTotal Internal Reflection Fluorescence (TIRF). Optical detection maycomprise the use of HiLo microscopy, for example as provided by Tokunagaet al (2008. Highly inclined thin illumination enables clearsingle-molecule imaging in cells. Nat Meth 5, 159-161). Opticaldetection may comprise the use of other glancing-incidence illuminationtechniques. Any suitable optical detection means may be used to detectoptical signals/emission in the membrane region immediately surroundingthe membrane and channel molecules of the membrane. Optical detectionmay comprise the use of surface plasmon resonance. Optical detection maycomprise the use of super-resolution microscopy, such as deterministicsuper-resolution, including STED, GSD, RESOLFT or SSIM; or stochasticalsuper-resolution, including SOFI, or single-molecule localizationmethods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM or dSTORM.Optical detection may comprise the use of epifluorescence microscopy,confocal laser scanning microscopy (LSM), or total internal reflectionfluorescence (TIRF) microscopy. Optical detection may comprise the useof fluorescence correlation spectroscopy (FCS). Image correlationspectroscopy (ICS) may be used to calculate the spatial correlationfunction of the fluctuations in fluorescence intensity of an image,which can be acquired by confocal or two-photon LSM or with TIRFmicroscopy. Optical detection techniques may be described in Ana J.Garcia-Sáez, Petra Schwille. Surface analysis of membrane dynamicsBiochimica et Biophysica Acta 1798 (2010) 766-776, the content of whichis incorporated by reference.

The method may comprise the detection of analyte interaction withmultiple channel molecules held in a membrane. The method may comprisethe detection of analyte interaction with multiple channel moleculesheld in multiple membranes. The method may comprise the detection ofanalyte interaction with one or more separate channel molecules held inmultiple membranes.

The method may comprise the detection of analyte interaction withmultiple channel molecules held in an array of membranes. The opticalfield of the optical detection means may be capable of encompassing anddetecting optical signals/emissions across an array of membranes andchannel molecules.

In an embodiment comprising the detection of analyte interaction withmultiple channel molecules held in multiple membranes, such as an arrayof membranes, the membrane potential may be provide by a single means.For example a single set of electrodes may be applied to provide themembrane potential for all membranes. For example, all membranes in anarray may be provided with a single cathode and single anode forproviding membrane potential. In an embodiment comprising the detectionof analyte interaction with multiple channel molecules held in multiplemembranes, such as an array of membranes, the membrane potential may notbe provide by multiple sets of electrodes, for example multiple cathodesor anodes.

Advantageously, the invention can be applied without the need formultiple electrode connections, which is a major issue holding back thefeasibility of high-throughput methods using channel molecules,membranes and optical detections means. The present invention overcomessuch an issue by the ability to apply a single pair of electrodes whichact upon all, or groups of, membranes in the method, leading to abreakthrough in the ability to provide high-throughput methodologies tooptical channel molecule detection technology.

The optical detection may be recorded by a charge-coupled device (CCD)camera. The optical detection may be recorded by an electron multiplyingCCD camera. Up to, and over, 2500 pores may be recorded simultaneouslywith an Electron Multiplying CCD camera (ixon3, Andor).

The term “at least partial blockage” of the channel molecule may beconsidered to be the reduction of flux of the signal molecule throughthe channel molecule due to the bulk of the analyte molecule. Theblockage may be complete blockage such that no flux of the signalmolecule can occur in the presence of the analyte in the channelmolecule. Partial blockage may allow for some signal molecules to passthrough the channel molecule.

The term “flux” may be considered to be the passage of a molecule fromone side of a membrane to the other side of the membrane through achannel molecule.

The term “optical signal” or “optical emission” may be considered to bethe emission of an energy, such as a wavelength, capable of beingdetected optically. For example fluorescence and light may be opticalsignals/emissions.

According to another aspect of the invention, there is provided abilayer array comprising:

-   -   opposing hydrogel surfaces provided by a first component        comprising an array of discrete hydrogel surfaces opposing a        hydrogel surface of a second component;    -   wherein the opposing hydrogel surfaces are immersed in a        hydrophobic medium comprising amphipathic molecules, and wherein        the opposing hydrogel surfaces are spaced apart sufficiently to        provide bilayers of the amphipathic molecules formed        therebetween;    -   channel molecules in the bilayers;    -   a means arranged to provide a membrane potential across the        bilayers.

The bilayer array of the present invention provides improvements overthe known bilayer methods, such as droplet-hydrogel bilayers (DHBs) andcan be used with the method of the invention herein. A DHB is a lowcost, simple and convenient system for basic biological sensing withoptical recording. However, a single large DHB (˜1 mm²) may lead to anunstable bilayer at high potentials, which can limit the scalability ofthe system. Besides that, only one type of analyte may be measured in asingle DHB at a time, which prohibits its application of biologicalscreening unless a highly parallel array of droplets and electrodes areused simultaneously. On the contrary, the present invention can beprovided in the format of a miniaturized, chip shaped and portabledevice containing a massive array of small but durable bilayers, whichis more ideal for biological sensing in ultra large throughput. Thischip device can be made of ion conductive hydrogel materials, whichelectrically connects all the bilayer with one pair of common electrodesto drive ion flux for optical recording. The bilayer array can localizebiological samples in position and each bilayer compartment requiresonly a minimum amount of precious biological samples for high throughputscreening.

The bilayer array may be used in the method for detection of analyteinteraction with a channel molecule held in a bilayer, according to theinvention herein.

The array of hydrogel surfaces may be provided on an array of pillarsextending from the first component. The first component may comprise anarray of pillars. The pillars may comprise and/or be formed of ahydrogel. The apex/top surface of the pillars may form the hydrogelsurface for forming the bilayer.

The pillars may be substantially square in cross-section. The pillarsmay not be circular in cross section.

A discrete hydrogel surface of the first component may be less thanabout 200 microns in diameter. A discrete hydrogel surface of the firstcomponent may be about 100 microns in diameter, or less. A discretehydrogel surface of the first component may be about 50 microns indiameter, or less. A discrete hydrogel surface of the first componentmay be about 20 microns in diameter, or less. A discrete hydrogelsurface of the first component may be less than about 40 mm² in surfacearea. A discrete hydrogel surface of the first component may be about 10mm² in surface area, or less. A discrete hydrogel surface of the firstcomponent may be about 2.5 mm² in surface area, or less. A discretehydrogel surface of the first component may be about 0.4 mm² in surfacearea, or less. Reference to the diameter or area of a discrete hydrogelsurface of the first component may refer to each hydrogel surface of thearray of hydrogel surfaces.

The total bilayer area provided by the array of bilayers may be at least1 mm². The total bilayer area provided by the array of bilayers may beat least 2 mm², at least 5 mm², at least 10 mm², at least 15 mm², atleast 20 mm², or at least 25 mm².

The array of discrete hydrogel surfaces of the first component may bearranged in an array of rows and columns, for example a gridarrangement. The array may comprise at least 2 discrete hydrogelsurfaces. The array may comprise at least 4 discrete hydrogel surfaces.The array may comprise at least 10 discrete hydrogel surfaces. The arraymay comprise at least 25 discrete hydrogel surfaces. The array maycomprise at least 100 discrete hydrogel surfaces. The array may comprisebetween about 2 and about 1000 discrete hydrogel surfaces. The array maycomprise between about 2 and about 2500 discrete hydrogel surfaces. Thearray may comprise between about 4 and about 2500 discrete hydrogelsurfaces.

The bilayers may be arranged in an array of rows and columns, forexample a grid arrangement. The bilayer array may comprise at least 10discrete bilayers. The bilayer array may comprise at least 50 discretebilayers. The bilayer array may comprise at least 100 discrete bilayers.The bilayer array may comprise at least 200 discrete bilayers. Thebilayer array may comprise at least 300 discrete bilayers. The bilayerarray may comprise at least 500 discrete bilayers. The bilayer array maycomprise at least 1000 discrete bilayers. The bilayer array may compriseat least 1500 discrete bilayers. The bilayer array may comprise at least2500 discrete bilayers. The bilayer array may comprise between about 10and about 3000 discrete bilayers. The bilayer array may comprise betweenabout 50 and about 10000 discrete bilayers. The bilayer array maycomprise between about 50 and about 10⁶ discrete bilayers. The bilayerarray may comprise between about 1000 and about 10⁶ discrete bilayers.

The first component may comprise or consist of a hydrogel. The firstcomponent comprising an array of hydrogel surfaces may be a single castof hydrogel.

The first component comprising an array of hydrogel surfaces may furthercomprise a barrier material arranged to prevent cross-contamination ofreagents and/or channel molecules between adjacent hydrogel surfaces.The barrier material may be arranged between the hydrogel surfaces, forexample between the pillars. The barrier material may be a solid. Thebarrier material may be solid and rigid. The barrier material may betransparent. The barrier material may be liquid impermeable. The barriermaterial may be non-porous. The barrier material may comprise or consistof a plastic polymer, such as poly(methyl methacrylate (PMMA). Thebarrier material may comprise or consist of any suitable material thatcan prevent translocation of chemical reagents, such as small molecules(e.g. fluorophores) and/or biological molecules, such as nucleic acid,peptides, or proteins (e.g. channel molecules or analytes). The barriermaterial may comprise or consist of non-conducting material. The barriermaterial may comprise materials selected from the group comprising PTFE;hydrophilic-treated PDMS; silica; glass; diamond; and acetal resin(Delrin™); or combinations thereof.

The first component may be circular in shape. The first component may bea disc shape. The first component may be sized to fit onto a standardmicroscope slide. The first component may be about 4-16 mm in diameter,such as about 8mm in diameter. The first component may be between about100 and about 1000 microns in thickness.

The first component may be supported by a plate. For example the firstcomponent may be in the form of a hydrogel on a backing plate. The platemay be a rigid solid. The plate may be transparent. The plate may beglass, such as a glass coverslip. The plate may be between 80 and about170 microns in thickness, or less. The plate may be positioned on thesurface of the first component opposite the hydrogel/bilayer-formingsurfaces.

Advantageously, providing the first component with a plate canfacilitate manipulation of a potentially weak and thin hydrogel basedfirst component. For example tweezers may be used to manipulate thefirst component during assembly of the bilayer array. The plate may alsobenefit the casting of a smooth, flat/planar surface on the firstcomponent.

The second component may comprise hydrogel, such as hydrogel layer. Thesecond component may comprise a planar hydrogel layer.

The second component may be sized to substantially match the size of thefirst component. The second component may be at least 4 mm in diameter,or about 8 mm in diameter. The second component may be about 5-100 mm²in size. The second component may be capable of supporting an array ofat least 4 bilayers, at least 10 bilayers, or at least 50 bilayers. Thesecond component may be about 0.1 microns in thickness. The secondcomponent may be less than about 10000 microns in thickness. The secondcomponent may be between about 0.01 and about 10000 microns inthickness.

The second component may comprise a plate. The hydrogel of the secondcomponent may be supported on a plate. For example the second componentmay be in the form of a hydrogel layered on a plate. The plate may be arigid solid. The plate may be transparent. The plate may be glass, suchas a glass coverslip. The plate may be between 80 and about 1000 micronsin thickness, or less. The combined thickness of the second componentwith the plate may be less than about 150 microns.

The means for providing a membrane potential may comprise cathode andanode electrodes. The cathode and anode electrodes may be arranged onopposing components, on either side of the bilayer in order to provide amembrane potential across the bilayer. The bilayer array may compriseonly a single set of anodes and cathodes for providing membranepotential across all the bilayers in the array. For example, eachseparate bilayer may not comprise a separate electrode connection. Acathode may connect to the first component, and an opposing anode mayconnect to the second component; or vice versa.

The hydrogel may comprise or consist of hydrophilic material. Thehydrogel may comprise or consist of hydrophilic polymer. The hydrogelmay comprise or consist of substantially transparent hydrophilicpolymer. The hydrogel may comprise or consist of agarose. Other hydrogelmaterials may be suitable, such as polyacrylamide, cross-linkedpolyethylene glycol, or nitro-cellulose.

The hydrogel may comprise or consist of less than 5% (w/v) agarose. Thehydrogel may comprise or consist of less than 4% (w/v) agarose. Thehydrogel may comprise or consist of about 3% (w/v) agarose. The hydrogelmay comprise or consist of greater than 1% (w/v) agarose. The hydrogelmay comprise or consist of 2% (w/v) agarose, or more. The hydrogel maycomprise or consist of between about 2% and about 4% agarose. Thehydrogel may comprise or consist of between about 2.5% (w/v) and about3.5% (w/v) agarose.

The amphipathic molecules may comprise or consist of lipid. The bilayermay comprise or consist of amphipathic molecules, such as lipids. Thebilayer may be a lipid-bilayer. The amphipathic molecules used in anymethod of the invention may be lipid molecules, in particular,surfactant molecules may be used. The lipid molecules may be selectedfrom the group comprising fatty acyls, glycerolipids,glycerophospholipids, sphingolipids, sterol lipids, prenollipids,saccharolipids, polyketides, phospholipids, glycolipids and cholesterol.

The lipid may include any of the group comprising monoolein;1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC);1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC);1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE);1-palmitoyl-2-oleoyl-phosphatidylethanolamine; and1-palmitoyl-2-oleoylphosphatidylglycerol (POPE/POPG) mixtures; ormixtures thereof.

The hydrophobic medium may comprise oil. The hydrophobic mediumcomprising amphipathic molecules may comprise or consist oflipid-in-oil. The oil may be a hydrocarbon, which may be branched orunbranched, and may be substituted or unsubstituted. For example, thehydrocarbon may have from 5 to 20 carbon atoms, more preferably from 10to 17 carbon atoms. Suitable oils include alkanes or alkenes, such ashexadecane, decane, pentane or squalene, or fluorinated oils, orsilicone based oils, or carbon tetrachloride; or mixtures thereof. Inone embodiment the oil is an n-alkane, such as a C10 to C17 n-alkane,e.g. n-hexadecane (C16). The oil may comprise a 1:1 (v:v) mixture ofhexadecane and silicone oil AR20 (Sigma-Aldrich).

In an embodiment comprising the use of a lipid-in-oil, the lipid-in-oilsolution may comprise from about 1 mg/ml to about 30 mg/ml of lipid inthe oil. The lipid-in-oil solution may comprise about 5 mg/ml of lipid.

The lipid may comprise a phospholipid, such as a phosphocholine lipid,e.g. 1,2-diphytanoyl-sn-glycero-3-phophocholine (DPhPC).

The composition of the hydrogel may be controlled to contain the correctsalts to allow an electrical current to be carried, for example, NaCl,KCl, MgCl₂ and/or other salts may be included. The hydrogel may alsocomprise common buffering agents to control pH, for example, Bis-tris,Tris, Hepes, sodium phosphate and/or potassium phosphate. The hydrogelof the first component may comprise a different composition to thehydrogel of the second component. For example, the bilayer may comprisea cis side and a trans side, wherein an appropriate composition isprovided on either side of the bilayer. The first component hydrogel maycomprise a potassium chloride buffer (for example, 1.5 M KCl, 10 mMHEPES, PH 7.0) and the second component hydrogel may comprise a calciumchloride buffer (for example, 0.75 M CaCl₂, 10 mM HEPES, PH 7.0). Thebuffer, for example on the cis side of the bilayer, may further compriseEthylenediaminetetraacetic acid (EDTA) for competitive binding of theion, such as

Ca²⁺, whereby fluorescence diminishes when away from the centre of thechannel due to the competitive binding.

Salts may also be included for other reasons, for example, to stabiliseproteins, to control binding components, to control the osmotic gradientacross the bilayer and/or to activate fluorescent probes.

According to another aspect of the present invention, there is provideda method of manufacturing a bilayer array comprising

-   -   providing a mould for casting a hydrogel first component,        wherein the mould comprises an array of cavities for forming the        hydrogel first component with an array of hydrogel pillars;    -   pouring a liquid arranged to set into a hydrogel into the mould;    -   laying a plate onto the surface of the liquid and setting the        liquid to form the hydrogel first component;    -   removing the mould from the hydrogel first component;    -   optionally coating a barrier material on the hydrogel first        component, between the hydrogel pillars;    -   adding a reagents and/or samples to one or more hydrogel        pillars;    -   providing a second component comprising a hydrogel surface on a        plate;    -   submerging the hydrogel surfaces of the first and second        components in a volume of hydrophobic medium comprising        amphipathic molecules, thereby forming a monolayer of        amphipathic molecules on the hydrogel surfaces of the first and        second components;    -   bringing the first and second components into an opposing        position relative to each other such that the monolayers of        amphipathic molecules form a bilayer.

According to another aspect of the present invention, there is provideda bilayer array formed by the method of the invention herein.

According to another aspect of the present invention, there is provideda method of manufacturing a component for a bilayer array comprising:

-   -   providing a mould for casting a hydrogel component, wherein the        mould comprises an array of cavities for casting a hydrogel        component comprising an array of pillars;    -   pouring a liquid arranged to set into a hydrogel into the mould;    -   laying a plate onto the surface of the liquid and setting the        liquid to form a hydrogel component;    -   removing the mould from the hydrogel component.

The method of manufacture may further comprise coating a barriermaterial onto the hydrogel component, between the pillars.

The array of cavities of the mould may be arranged in an array. Thecavities may be arrayed in rows and columns. The array of cavities maybe arranged in a grid.

Coating the barrier material on the hydrogel first component maycomprise spinning the coating in a liquid form, and allowing the liquidform of the barrier material to set.

The mould may be etched by photolithography. The mould may be etchedfrom a photoresist material, for example SU-8 or similar.

According to another aspect of the present invention, there is provideda component for a bilayer array formed by the method of the inventionherein.

According to another aspect of the present invention, there is provideda hydrogel component for a bilayer array comprising an array of pillarsextending from the surface of the hydrogel component, and a barriermaterial coating the surface of the component between the pillars.

According to another aspect of the present invention, there is provideda kit for forming a bilayer array comprising:

-   -   a first component comprising an array of discrete hydrogel        surfaces; and    -   a second component comprising a hydrogel layer.

The kit may further comprise a volume of hydrophobic medium comprisingamphipathic molecules. The kit may further comprise a cathode and anode.The kit may further comprise channel molecules. The kit may furthercomprise reagents. The kit may further comprise one or more buffers.

According to another aspect of the present invention, there is provideda kit for manufacturing a bilayer array comprising:

-   -   a first mould for casting a first component comprising an array        of discrete hydrogel surfaces; and    -   a second mould for casting a second component comprising a        hydrogel layer.

The kit(s) of the invention may further comprise instructions formanufacture and/or assembly and/or use of the bilayer array.

The kit for manufacturing a bilayer array may comprise a hydrogelmaterial in liquid form. The kit for manufacturing a bilayer array maycomprise a hydrogel material in a powder or granular form, which iscapable of being reconstituted in an aqueous medium. The kit formanufacturing a bilayer array may comprise a hydrogel material in asolid or gel solid form, which can be melted/liquified for casting intothe mould. The kit for manufacturing a bilayer array may comprise abarrier material. The kit for manufacturing a bilayer array may furthercomprise a volume of hydrophobic medium comprising amphipathicmolecules.

The kit for manufacturing a bilayer array may comprise a cathode andanode. The kit for manufacturing a bilayer array may comprise channelmolecules. The kit for manufacturing a bilayer array may comprisereagents. The kit for manufacturing a bilayer array may comprise one ormore buffers.

According to another aspect of the present invention, there is provideda method of optical screening of a polynucleotide present in one or moresamples, the method comprising the steps:

-   -   providing a membrane comprising channel molecules held in the        membrane;    -   providing one or more template polynucleotides and one or more        polynucleotides from one or more samples on one side of the        membrane;    -   providing a membrane potential across the membrane, such that it        provides flux of a signal molecule through the channel        molecules;    -   detecting hybridisation, or lack thereof, of the one or more        template polynucleotides with polynucleotide in the one or more        samples by optically detecting any modification in flux of the        signal molecule through the channel molecules.

The method of optical screening may comprise the simultaneous orparallel screening of multiple samples and/or may comprise the use ofmultiple template nucleotides. For example, the method of opticalscreening may comprise the use of the bilayer array according toinvention, wherein the bilayer array comprises an array of bilayerscomprising the channel molecules held in the bilayers. The one or moretemplate polynucleotides may be deposited on the one or more hydrogelsurfaces of the first component, and the polynucleotides from one ormore samples may be deposited on the one or more hydrogel surfaces ofthe first component.

The same template nucleic acid sequence may be provided on two or more,or each, hydrogel surfaces of the first component. The same samplenucleic acids may be provided on two or more, or each, hydrogel surfacesof the first component. A different template nucleic acid sequence maybe provided on two or more, or each, hydrogel surfaces of the firstcomponent. Different sample nucleic acids may be provided on two ormore, or each, hydrogel surfaces of the first component.

According to another aspect of the present invention, there is providedthe use of the bilayer array of the invention herein, for opticalsequencing, and optionally wherein the optical sequencing is parallelsequencing in an array.

According to another aspect of the present invention, there is providedthe use of the bilayer array of the invention herein, for analyteanalysis, and optionally wherein the analyte analysis is parallelsequencing in an array.

According to another aspect of the invention, there is provided a methodfor detection of analyte interaction with a nanopore held in a bilayerof amphipathic molecules, comprising:

-   -   providing a bilayer of amphipathic molecules, wherein the        bilayer comprises one or more nanopores;    -   providing a first signal-associated molecule on one side of the        bilayer, wherein the first signal-associated molecule is capable        of flux through the nanopore(s) by the action of a membrane        potential across the bilayer;    -   providing a second signal-associated molecule on the opposing        side of the bilayer relative to the first signal-associated        molecule, wherein the first and/or second signal associated        molecules are arranged to emit an optical signal when in        contact;    -   providing an analyte on at least one side of the bilayer;    -   applying a membrane potential across the bilayer such that the        first signal-associated molecule is transported through the        nanopore and interacts with the second signal-associated        molecule to emit an optical signal;    -   detecting the optical signal; and    -   detecting any modification, or lack thereof, in the optical        signal as the flux of the first signal-associated molecule is        modified by at least partial blocking of the nanopore by the        analyte as it interacts with the nanopore.

The method of the invention herein, and/or the bilayer array of theinvention may be manipulated or implemented by a robotic system. Forexample, the deposition of sample, analyte, channel molecules and/orother reagents onto the hydrogel, or into the region of the membrane maybe carried out by robot spotting, or robot pipetting. A droplet ofsample, analyte, channel molecules and/or other reagents may be providedto the robot, which may spot the sample, analyte, channel moleculesand/or other reagents in the bilayer array. Once the first component andsecond component of the bilayer array is formed, the use of the bilayerarray may be fully or partially automated.

Advantageously, the use of an automated robot is made possible by abilayer array of the present invention which allows individual spots ofsample, analyte, channel molecules and/or other reagents to be depositedon the discrete hydrogel surfaces prior to forming the bilayers.Automated robot manipulation greatly enhances the use of the method ofthe invention and the bilayer array of the invention for high-throughputscreening, analysis and sequencing.

The skilled person will understand that optional features of oneembodiment or aspect of the invention may be applicable, whereappropriate, to other embodiments or aspects of the invention.

Embodiments of the invention will now be described in more detail, byway of example only, with reference to the accompanying drawings.

FIG. 1 | Evaluating the amplitude resolution of optical single channelrecording by static DNA blockages in DHB. a, The diagram of a single DHBin the 16 well measurement device (FIG. 5). A 60× TIRF objective is usedboth for TIRF illumination and image recording. Voltage protocols areapplied with a pair of Ag/AgCl electrodes as illustrated in the figure.Agarose is coated on the ground electrode to facilitate electrodestabbing into the droplet. b, The diagram of the area near the bilayer.Free αHL in the droplet spontaneously inserts into the DHB. At +100 mV,the area near an inserted αHL shows strong fluorescence when Ca²⁺ isdriven through the nanopore and bind with Fluo-8. At this potential,streptavidine (square) tethered ssDNA (line/curves) is also electricallydriven into the pore, which blocks the Ca²⁺ flow and reduces thefluorescence. c, The fluorescent trace of a representative DNA blockagecycle. The trace amplitude is normalized so that the mean intensities of(III) and (IV) are 0 and 1. The normalized fluorescent amplitude of (II)identifies the DNA being captured. d, A frame containing fluorescentspots representing open (circles A) and blocked nanopores (circles B) toshow the throughput of parallel recording. Scale bar: 10 μm. e, Acontinuous trace of nanopore blockages with a mixture of 2 types of DNA(X₅, cyan, histogram level 3; C₄₀, blue, histogram level 4). Level 6 inthe histogram is due to the gating of αHL at negative potential. f, Thecorrelation between optical recording (Table 2) and patch clampelectrical recording (Table 3) for different DNA blockage activities.

FIG. 2 | Demonstrating the time resolution of optical single channelrecording by kinetic miRNA unzipping in DHB. a, A representative miRNAunzipping event in the nanopore with optical recording. The miRNA ishybridized with the DNA probe. Poly-C ssDNA tags on both ends of theprobe are designed to initiate unzipping. At +160 mV, an open nanopore(I) shows strong fluorescence and then the hybridized nucleic acidcomplex is captured to unzip in the pore (II). Upon finishing unzipping,the DNA probe has translocated through the pore and the miRNA is leftbehind in the vestibule (III). Then the miRNA translocates through thepore (IV) and the pore re-opens (V) for the next event. b,Characteristic events for different probe/miRNA combinations and themiRNA controls. Matched/Un-matched miRNA to the probe normally generateslong/short T1 events while miRNA translocation doesn't show T1 event atall. c, A continuous fluorescent trace of miRNA (Plet7a/Let7a) unzippingat +160 mV. Magenta fitting lines highlight the T1 events. d, Thestatistics of mean duration time for different probe/miRNA combinationsand the miRNA controls. The matched probe/miRNA shows significantlylonger T1 than the unmatched counterpart, which further confirms that Tiis hybridization strength dependent. e, The histogram of T1 for all theprobe/miRNA combinations. The distribution follows an exponentialfitting and the fitted rate constant reflects the hybridization strength(Supplementary Discussion 4).

FIG. 3 | Optical single channel recording in a multiplexed HHBa chip. a,A casted hydrogel chip, scale bar: 4 mm (FIG. 20). The image inset showsan array of micro-pillar patterns on the chip surface, scale bar: 140μm. b, The diagram of a complete HHBa measurement procedure. Biologicalsamples (αHL) are spotted onto the pillars (FIG. 22) and then the chipis flipped over to form HHBa with the substrate agarose in lipid/oil(FIG. 21). c, Bilayer array detachment. To show the edge of thebilayers, after HHBa formation, the chip is lifted gradually to detachthe bilayers. Clear boundary lines between formed and un-formed bilayerscan be visualized (white arrows). Scale bar: 140 μm. d, The intensitychange of the fluorescent spots in the HHBa with +/− applied potential.αHL is spotted onto specific pillars (arrow 1). The inserted pores showstrong/weak fluorescence spots at +/−50 mV. Each spot represents asingle inserted nanopore. Scale bar: 35 μm. The fluorescent image isbackground normalized for optimized image contrast (FIG. 23). e, Thefluorescent trace of the areas in d (pointed by arrows 1 and 2) withvoltage protocols. A nanopore shows on/off fluorescent levels with +/−50mV applied potential. The un-spotted HHB (purple arrow) shows no poreinsertions and the fluorescence stays constant with voltage protocols.

FIG. 4 | Parallel biological sensing in HHBa. a, Stitching multiplefluorescence images (FIG. 24) to show a larger area of the chip withmultiple types of biological samples (1. −αHL, −DNA; 2. +αHL, −DNA; 3.+αHL, +DNA). A single frame (the yellow/white dotted square)accommodates 4 bilayers at a time. Scale bar: 40 μm. b, The diagram ofthe HHBa measurement in TIRF mode. By analyzing the fluorescentintensity of each spot, each HHB could perform an independent nanoporemeasurement in single molecule. c, Analyzing single molecule nanoporeactivities in parallel. The fluorescent traces are simultaneouslyrecorded from nanopores in different bilayers of the array (a,yellow/white square). The unspotted HHB shows constant fluorescence(Trace 1). However, the HHB loaded with αHL shows synchronizedfluorescence change with voltage protocols (Trace 2). DNA (C₄₀) blockageevents are only detected in Trace 3.

FIG. 5 | The DHB measurement device. The device is manufactured asreported before8 with a CNC milling machine (Modela MDX-40, Roland).a-c, The standard three-view drawing of the DHB device used in theexperiment. Scale unit: mm. The agarose (0.75% low melt agarose) coatedcoverslip sticks to the bottom of the plastic plate when molten agarose(2.5% low melt agarose) is filled into the device (b). d, The actual DHBmeasurement chamber as demonstrated in (a-c). Inlet (arrow A)/Outlet(arrow B) holes on the device is designed to facilitate the filling ofmolten agarose. The extra hole on the outlet side helps air bubbles toescape during the filling. Each one of the 16 holes (central part of thedevice) accommodates a single DHB. However, only one DHB can bemonitored simultaneously.

FIG. 6 | The electrical measurement in the DHB. a, The schematic diagramof the electrical measurement in a DHB. Nanopores and streptavidintethered ssDNA (C40, 267 nM) are placed in the droplet. There aremultiple nanopores inserted in the DHB and the total ionic current isrecorded by the patch clamp amplifier. b, A representative electricaltrace of DNA blockages in DHB. At +100 mV, the current transientlyachieves ˜1500 pA. Then, streptavidin tethered ssDNA in the dropletstarts blocking each pore sequentially, which appears as a step shapedtrace in the current. c, The histogram of the electrical trace in (b),The separations between the adjacent peaks (82.9±6.5 pA, N=12) iscomparable to the typical DNA blockage amplitude in a PLM measurement.

FIG. 7 | Current-Voltage (IV) curve. The IV curve of a single nanoporeis measured in the PLM. IV measurements for all four buffer combinationsin Cis and Trans chambers are recorded. a, The schematic diagram of IVcurve measurement with PLM. The Cis side is defined as the chamber withnanopores and is electrically grounded. To compare the conductancechange of the same nanopore with different buffer combinations, thebuffer in either the Cis or the Trans side are exchanged withoutbreaking the bilayer. b, The IV curve measurement with all fourdifferent combinations of calcium chloride (0.66 M CaCl2, 8.8 mM HEPES,Ph 7.0) and potassium chloride (1.32 M KCl, 8.8 mM HEPES, Ph 7.0)buffer. Please note that at positive potential the conductance of asingle pore is lower (as pointed out by the arrow) when the calciumchloride buffer is placed on the Trans side.

FIG. 8 | Fluorescence-Voltage (FV) curve. In OPC, the fluorescentintensity, which is proportional to the Ca2+ flow rate, is modulated bythe applied potential. a, The fluorescence of a single nanopore atdifferent applied potentials. The fluorescent intensity gradually dropswith the potential ramp from +100 mV to −100 mV. Selected image frameson the right demonstrate the change of the spot brightness at differentpotentials. b, The applied voltage protocols. c, The FV curve of an opennanopore. Linear curve fitting (y) is performed for the FV curve atpositive potentials and exponential curve fitting (x) is performed atnegative potentials.

FIG. 9 | 2D Gaussian fitting and position tracking. The demonstrateddata is based on the OPC of a single nanopore in DHB. a, The fluorescenttrace containing 2 types of DNA blockage events. b-f, Raw image framesof a single fluorescent spot aligned with the trace. g-k, The 2DGaussian fitting result of the raw images in (b-f). The colour coding ofthe cone, which is consistent with the fluorescent intensity, helps toresolve subtle differences in the residual fluorescence of C40 (i) andX5 (j) blockage events. The position tracking based on 2D Gaussianfitting. The position of the pore is defined as the centre of the fittedcone. Here the tracking result from 100 frames is demonstrated. Thestandard deviation on the x direction is 11.33 nm and on the y directionis 13.83 nm.

FIG. 10 | The demonstration of parallel recording. Multiple nanoporescan be tracked and recorded simultaneously. a, The superimposed imagefrom 1000 frames containing 3 nanopores. The position trackings aremarked with colour circles (pore 1: yellow; pore 2: pink; pore 3: cyan).Pore 3 (cyan), which is forced to drift by the edge of the bilayer,generates a blurry tail from the tracking (arrow) in the superimposedimage. Scale bar: 10 μm. b, The tracking result of the 3 pores. Thetrack of pore 3 shows a stretched line due to the pore drifting. c-h,Selected image frames of pore activities from the 3 pores. Cartoonschematics on top of each frame demonstrate the independent poreactivities from the 3 pores. Streptavidine tethered C40 is placed in thedroplet to block the pore. Scale bar: 10 μm. i-k, Independentfluorescent traces from the 3 pores in the same field of view.

FIG. 11 | The minimum spatial resolution with optical single channelrecording. In principle, fluorescent spots which are separated with morethan their half height width should be resolved. To test the minimumspatial resolution with the bilayer, the agarose coating below the DHBis over hydrated to increase the mobility of the bilayer. Pores driftstochastically within the DHB and generates various pore to poredistance with time. The tracking resolution is thus estimated byanalyzing pore pairs with tiny separations. a, The image framecontaining many nanopores. The tracking is marked by colour codedcircles on the spots. Scale bar: 15 b, This image demonstrates theminimum pore to pore distance that the tracking algorithm can stilldistinguish. The pore to pore separation is 3.2 μm.

FIG. 12 | DNA blockages in the fluorescent traces. Three fluorescenttraces are demonstrated to show different types of DNA blockages in DHB.a, The representative fluorescent trace of X5 DNA blockages. b, Therepresentative fluorescent trace of X3 DNA blockages. c, Therepresentative fluorescent trace of C40 DNA blockages.

FIG. 13 | Simultaneous recordings of 12 pores. The images are recordedin DHB. a-d, Selected image frames containing the colour coded trackingmarkers over 12 fluorescent spots. Solid arrows point to open pores thatare blocked (as pointed by the dashed colour arrows) in the next frame.Potential: +100 mV. Scale bar: 15 μm. e, The image frame at −50 mV. f,The image frame at 0 mV. g-j, 4 selected fluorescent traces with DNA(C40) blockage events (blue arrows).

FIG. 14 | The histogram of the residual current. The experiment isperformed with a single nanopore in PLM. Three types of DNA(streptavidine tethered C40, X3 and X5) are mixed with the sameconcentration (89 nM each). ssDNA with a basic nucleotides replacementsin sequence may have an advantage in captured rate due to the reducedmolecular weight and the increased mobility. a, The histogram based on1000 DNA blockage cycles. b, the corresponding Gaussian fitting result.

FIG. 15 | Electroporations at high potentials. Electroporations, whichare non-specific holes in the lipid membrane, generate huge ion leakageand may rupture the bilayer. In a single large DHB (>1 mm2), highapplied potential (>160 mV) normally leads to electroporation formation.The bilayer stability against high potentials is reduced by increasingsize of a single bilayer. a-e, Electroporation formation process at highpotentials. Electroporations are recognized as bright spots withabnormal sizes and vigorous intensity fluctuations. On the contrary, aproperly inserted αHL shows consistent and stable fluorescent levels.The yellow/white dashed line represents the boundary of the DHB. Scalebar: 20 μm. f, The DHB is irreversibly ruptured by high potentials,which generates extremely strong fluorescence due to abundant Ca²⁺ andFluo-8 binding.

FIG. 16 | The fitting algorithm for miRNA unzipping traces. Thefluorescent trace is recorded in DHB with +160 mV potential. Althoughthe minimum exposure time for a single frame in OPC is 3 ms for now. Afitting algorithm is developed to break this time resolution evenfurther. In general, sharp transitions in the fluorescent trace appearas peaks in the 1st order derivative result. By 2D Gaussian fitting ofthese peaks, the accurate transition time between states could beestimated from the fitted centre. a, A representative miRNA unzippingcycle. b, The 1st order derivative of the trace in a. The fitted peaksdetermine the accurate transition time (dash lines) between molecularstates. c, The peaks in the 1st order derivative trace and the fitting.d, Automated event selection. A typical miRNA unzipping event has 3characteristic state levels (FIG. 2a ). By analyzing the amplitude andthe transition time of the fluorescent trace, miRNA unzipping events canbe picked up automatically with a homemade labview program.Periodically, −50 mV is applied across the DHB to clear the pore fromclogging. Partial events (0-1 seconds in d) and events at incorrectpotentials (17.5-18.5 seconds in d) are not included for statistics.

FIG. 17 | miRNA unzipping in the 15 sec traces. The measurements aredone in DHB. All four combinations of matched/mismatched probe/miRNAhave been tested. In general, long T1 events (>1 sec) are onlydetectable when the miRNA is complementary to the probe. a,Plet7a/Let7a. b, Plet7a/Let7i. c, Plet7i/Let7i. d, Plet7i/Let7a.

FIG. 18 | Voltage dependence of miRNA unzipping for Plet7a/Let7i. At lowapplied potential (<120 mV), the duration time for T1 is normally toolong (T1>10 s) for sequence complementary probe/miRNA combinations andthe pore could be easily clogged by the hybridized sample withoutunzipping. Thus, a mismatched probe/miRNA sample (Plet7a/Let7i) is usedto demonstrate the voltage dependence from 100 mV to 160 mV. a, Thehistogram of T1 at different applied potentials. The fitted rateconstant in the distribution suggests that the unzipping is much fasterat higher potentials. b, The mean duration time of T1, T2 and T3 atdifferent potentials. The mean duration time of T1 is significantlylonger at +100 mV. c, The plot of the rate constant vs. voltage with anexponential fitting.

FIG. 19 | The HHBa measurement chamber. The device is manufactured witha CNC milling machine (Modela MDX-40, Roland). a-c, The standardthree-view drawing of the HHBa chamber used in the experiment. Scaleunit: mm. The agarose coated (0.2% low melt agarose) coverslip sticks tothe bottom of the device when molten agarose (2% low melt agarose) isfilled into the setup (b). d, The actual HHBa measurement chamber. Inlet(arrow A)/Outlet (arrow B) holes on the device is designed for moltenagarose to fill into the channel. The extra hole on the outlet sidehelps air bubbles to escape during the filling.

FIG. 20 | The hydrogel chip fabrication procedures. The hydrogel chip isfabricated according to the following procedures. Step a-c is performedaccording to the standard soft lithography protocol3. Step d-f issimilar to the fabrication of an agarose stamp9 and the PDMS mould canbe reused for multiple times. a, Patterning of SU-8 pillars with thestandard photolithography protocols (MicroChem). b, Casting PDMS wellswith the SU-8 mould (a). c, Peeling off the PDMS from the SU-8 mould. d,Filling the micro-cavities of the PDMS mould with molten agarose (3%(v/v) agarose for routine use, 1.32 M KCl, 8.8 mM HEPES, 8.8 mM EDTA, Ph7.0). e, Desiccator degassing. Air bubbles in the hydrogel chip needs tobe removed and a piece of coverslip is immediately placed on the back ofthe hydrogel. f, Gelling of the agarose at low temperatures (4° C., 30minutes). g, Peeling off the casted hydrogel chip from the PDMS mould.h, Spin coating (4000× rpm, 60 s) of PMMA 495/A5 (0.2% in anisole) abovethe hydrogel chip. Immediately after the spin coating, a PMMA film willform in the gaps between the pillars. i, Immersing the chip inhexadecane to avoid de-hydration. Fluo-8 dye (1 μg/μL) is fused into thehydrogel and the. It takes ˜4 hours to achieve a homogeneousdistribution of the dye. The chip immersed in hexadecane can be storedat room temperature for >48 hours. j, Comparing the size of the chipwith a 5 cent euro coin. Scale bar: 8 mm. The chip can be easilymanipulated with a pair of tweezers. Image inset: Bright fieldmicroscopic image of the hydrogel pillars on the chip. Scale bar: 140μm.

FIG. 21 | The electrode for the HHBa chip. A special electrode ring(Ag/AgCl) is designed to hold the chip and secures the electricalconnections. a, The top view diagram of the electrode. b, The side viewdiagram of the electrode with a chip. The chip is placed onto theelectrode by a pair of tweezers. The electrode is coated with a layer ofagarose to make the surface more hydrophilic so that the electrode andthe chip spontaneously form a tight electrical connection upon physicalcontact. c, The actual electrode. Scale bar: 5 mm. d, The diagram ofHHBa formation in 3 mM lipid/oil.

FIG. 22 | Programmed chip loading with a home-made spotting robot.Biological samples can be selectively loaded/spotted onto the surface ofthe pillars with a sharp capillary tip on a spotting robot (Patchstar,Scientifica), which loads samples (αHL or DNA in source droplets) andspots them onto the pillars of the chip by physical contact. Theloading/spotting is controlled by a labview program which enablesautomated and accurate positioning (up to 100 nm/step). a-d, Monitoringthe sequential spotting. When the spotting tip is in physical contactwith the pillar (a,d), the capillary apex is clearly visible as afocused spot. Scale bar: 210 μm. e-h, The cartoon diagram describing thespotting process consistent with the images above. The red colour at theend of the capillary represents the loaded biological samples.Biological samples are spotted onto the pillars by passive diffusionwhen the tip and the pillars are in physical contact. The spottingefficiency depends on various parameters (The initial sampleconcentration in the capillary; The spotting duration time; The diameterof the capillary tip) and is not particularly studied in this paper. Thearrows indicate the movement direction of the capillary in the followingstep. I, Multi-sample spotting. In general, different types ofbiological samples can be loaded/spotted onto the same chip from varioussource droplets for biological screening in single molecule.Multi-sample spotting is done in multiple steps.

FIG. 23 | Background normalization treatment. In the TIRF measurement ofHHBa with full frame, background normalization treatment is normallyneeded to enhance the image contrast for optimized printing quality.Note that the background normalization is not performed for anyquantitative data analysis and all the fluorescent traces beingdemonstrated in this study are extracted directly from the raw imagedata without any background treatment. a, A single frame from the rawimage series. Scale bar: 35 b, The background profile of a. The profileis generated by averaging 100 raw image frames followed with Gaussianfiltration (ImageJ) to smooth the sharp features of the fluorescentspots. c, The image frame after background normalization (FIG. 3d ). Thenormalization is done by dividing the raw image series with thebackground profile image (Image Calculator, ImageJ). To enhance thesignal noise ratio, 12 subsequent frames are superimposed (Z project,ImageJ). d, A single frame from another image series. Scale bar: 35 μm.e, The corresponding background profile of d. f, The image frame afterbackground normalization treatment.

FIG. 24 | Image stitching for expanded field of view. Due to the limitedFOV (150 μm×150 μm) for a 60× oil immersion TIRF objective, only part ofthe HHBa chip (4/2500 bilayers) can be monitored simultaneously. Todemonstrate an expanded area of the chip in TIRF mode, the objective ismoved over different areas during the recording and a minimum 100 framesare recorded for each area. A standard deviation image (SDI) iscalculated (Z-project, ImageJ) from each image series. Frequentintensity change of the fluorescent spots leads to large standarddeviations in the pixel values and appear as white spots in the SDI. TheSDI image treatment picks up specifically functioning nanopores and anyscattering spots (fluorescent spots due to dusts or satellite droplets)with constant fluorescence will be emitted automatically. This treatmentalso minimizes the contrast difference between adjacent images to bestitched (MosaicJ, Fiji). a-f, A series of SDI in full frame fordifferent parts of the array. Scale bar: 30 μm. g, The final stitchedimage from a-f. Unloaded HHBs are marked with arrows and no spots arevisible. Scale bar: 50 μn.

EXAMPLE Multiplexed Nucleic Acid Sensing Using an Optical NanoporeArray.

To evaluate the feasibility and resolution of optical sequencing, inthis study, prototypes of nucleic acid sensing with αHL nanopores aredemonstrated using total internal reflection fluorescence (TIRF)microscopy. In general, αHL nanopores, which are placed on the Cis sideof the bilayer, spontaneously insert into either a single droplethydrogel bilayer (DHB) (FIG. 1b ) or a hydrogel hydrogel bilayer array(HHBa) (FIG. 4b ) and conduct Ca²⁺ from the Trans side of the bilayerinto the Cis. Ca²⁺ binds with Fluo-8 in the Cis and emits fluorescence(517 nm) upon laser illuminations (473 nm). Clouds of Fluo-8/Ca²⁺ appearas bright spots centred on each pore (FIG. 1d ) and the fluorescencediminishes when away from the centre due to the competitive binding ofCa²⁺ with Ethylenediaminetetraacetic acid (EDTA) in the droplet or theHHBa chip. The average size of each spot at maximum intensity is around3 μm in diameter and in principle over 2500 pores can be recordedsimultaneously with an Electron Multiplying CCD camera (ixon3, Andor).The fluorescent intensity, which is proportional to the Ca²⁺ flow rate,is enhanced at higher potentials (FIG. 8). Analytes (DNA or RNA) in thenanopore reduces Ca²⁺ flow rate and the residual fluorescence (FIGS. 1c,2a ) provides accurate information (1 pA equivalent amplitude resolutionand 3 ms temporal resolution) for biological sensing.

To calibrate the amplitude resolution, a set of streptavdine tetheredssDNA with minor sequence differences (C₄₀, X₃ and X₅, Table 1) isdesigned as molecular rulers. Different lengths of a basic nucleotidesare replaced in the sequence according to the position of the 2^(nd)recognition site of αHL [Stoddart, 2009 PNAS]. To perform opticalrecording, a DHB is formed (FIG. 1a ) in 3 mM lipid/oil (SupplementaryMaterials) and nanopores in the droplet spontaneously insert into thebilayer.

At +100 mV, an open αHL in the DHB appears as a bright spot due to theabundant Ca²⁺ being transported. Immediately, streptavidine tetheredssDNA blocks the pore and reduces the fluorescence (FIG. 1b ) until theapplied potential is flipped to negative. The normalized mean amplitude(Table 2) in phase II identifies the DNA being trapped in the pore.Repeated measurement cycles form a continuous fluorescent trace (FIG. 1c) and the events are accumulated for statistics. Compared with theresults of patch clamp recording in a planar lipid membrane (PLM) (FIG.14), the residual fluorescence and the residual current shows a linearrelationship statistically (FIG. 1f ) and the residual currentseparation between X₃ and X₅ demonstrates that OPC resolves ˜1 pAequivalent resolution.

With up to ˜300 Hz frame rate and ˜1 pA amplitude resolution, opticaldetection should resolve fast kinetic process like miRNA unzipping innanopores. miRNA, a short (˜22 nucleotides) and non-coding RNA fragment,is of significant biological importance but difficult to bequantitatively analyzed by PCR based methods. The miRNA, when hybridizedwith a DNA probe and electrically stretched in a nanopore, can be forcedto unzip. The unzipping kinetics, which is recorded from a single porein the PLM, reveals the miRNA identity statistically [Wang, 2011 NatureNano]. However, the unzipping duration time is widely distributed andrequires a significant amount of events for statistics. Opticaldetection methods, which image pore activities in massive throughput andproduce streams of single molecule fluorescent traces simultaneously,are ideal for ultra fast recording and screening of miRNA samples.

As a proof of concept demonstration, DNA probes with C₃₀ tags (Plet7aand Plet7i) are designed to be sequence complimentary to their miRNAcounterpart (Let7a and Let7i). All four combinations of hybridized miRNAwith DNA probes are thermally annealed. A droplet (1.32 M KCL, 8.8 mMHEPES, 8.8 mM EDTA, Ph 7.0, 40 μM Fluo-8, 1.2 nM αHL) containing onetype of Probe/miRNA (267 nM) forms a DHB with the substrate agarose in 3mM lipid/oil as described in FIG. 1 a.

At +160 mV constant bias, the fluorescent spots on each αHL “blinks”spontaneously when miRNA unzips and translocates through the pores. Atypical unzipping event includes 3 blockage levels (FIG. 2a ) andappears repetitively at a constant bias (FIG. 2c ). Based on the opticalrecording results for all combinations of Probe/miRNA hybridizations and2 types of miRNA controls, matched/unmatched Probe/miRNA generateslong/short T1 duration time while T1 disappears in miRNA translocationevents (FIGS. 2b, d ). Thus, it is concluded that T1 reflects thehybridization strength between the miRNA to the probe and is analyzedfor miRNA identifications. T1 is widely distributed and can be fit by anexponential curve [Sauer-Budge, 2003 PhysRevLett]. The fitted rateconstant (Table 5) is used to distinguish the miRNA identity.

Agarose, a low cost and bio-compatible hydrogel material, is widely usedfor gel electrophoresis and can be casted with micro-features [Mayer,2004 Proteomics]. This hydrogel based chip device with micro-pillarstructures (FIG. 3a ) forms a HHBa (hydrogel-hydrogel bilayer array)with the substrate agarose in 3 mM lipid/oil (FIGS. 3b, 4b ).

Similar to a DHB, the HHBa can also be imaged by TIRF microscopy andeach individual bilayer performs independent single molecule sensing(FIG. 3c ). αHL can be selectively loaded into specific compartments ofthe HHBa. In a loaded bilayer, each inserted pore can be opticallyresolved as bright/dim fluorescent spots at +/−50 mV (FIG. 3d ).Fluorescent traces from αHL and unloaded area show distinguishablesignal patterns (FIG. 4e ), which suggests the application of parallelrecording of multiple samples in the same field of view for singlemolecule biological screening.

As demonstrated in the DHB system, streptavidine tethered ssDNA blocksαHL with unique blockage signals in the fluorescent traces. As a proofof concept experiment for biological screening, nanopore activitieswith/without DNA can be monitored simultaneously on the same HHBa chipin the same field of view. After spot loading and HHBa formation, αHL onthe surface of the pillar inserts into the bilayer (FIG. 4b ) and appearas bright fluorescent spots in loaded areas while the unloaded pillarsmaintain clean of spots (FIG. 4a ). At +160 mV, streptavidine tetheredC₄₀ blocks αHL and reduces fluorescent intensity of the spots. Singlemolecule fluorescent traces recorded simultaneously from HHBs withdifferent loaded samples show clear discriminations (FIG. 4c ) in thesame field of view.

Besides the potential applications for biological screening, the HHBachip also enlarges the total bilayer area (25 mm²) with improved bilayerstability (>200 mV can be applied).

In conclusion, high resolution optical detection methods (˜1 pA, ˜3 ms)which monitor ion flux similar to electrical recording fits a wide rangeof nucleic acid sensing in nanopores as demonstrated herein. Being ahighly parallel technology, the method is ideal for data intensivemeasurement, such as a nanopore array for human genome sequencing. With3 μm pore to pore separation, an ideal hexagonal array of ˜10⁶ nanoporesshould function in parallel within a ˜mm² area. A full human genomecould be sequenced in 15 minutes with this throughput according to thereported nanopore sequence speed. Equipped with a more advanced hydrogelchip device, the applications of the method is expanded to more generalsingle molecule biological screening. Technically, an OPC device doesn'trequire any high end electrical components as long as the voltage can beapplied with macroscopic common electrodes. The demonstrated device (DHBand HHBa) is also made of extremely low cost and accessible materials.These technical advantages enable a chip device for single moleculebiological sensing with extremely affordable price in a miniaturizedsize.

SUMMARY

By optically encoding the Ca²⁺ flux the detection of nucleic-acidbinding events in nanopores was parralellised. Parallel recordings at adensity of ˜10⁴ mm⁻² measurements in a single droplet hydrogel bilayer(DHB) have been demonstrated.

Both static DNA blockage and kinetic miRNA unzipping events can bemonitored optically for single molecule nucleic acid identifications.Sub-pA equivalent amplitude resolution and 3 ms temporal resolution isdemonstrated, which enables discrimination between nucleic acids with2-4 bases difference. To further expand this platform, hydrogel hydrogelbilayer array (HHBa) is formed with micropatterned hydrogel chip, whichis also compatible with a spotting robot for biological screeningapplications. Based on the enzymatic ratchet speed (˜35 Hz), thisoptical recording platform should produce sequencing signal with a rateof 10⁶ nucleotides mm⁻²s⁻¹, which paves the way to 15 minutes humangenome sequencing and other general applications of single moleculesensing with nanopores.

Methods:

DHB formation. αHL (1.2 nM) and analytes (streptavidine tethered ssDNAor miRNA) (267 nM) are placed in a 350 nL droplet (1.32 M KCL, 8.8 mMHEPES, 8.8 mM EDTA, Ph 7.0, 40 μM Fluo-8), which is incubated in 3 mM1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in oil(Supplementary Materials) to form an external lipid monolayer coating.The droplet is pipette transferred into the measurement chamber (FIG. 1a, FIG. 5), where a thin layer (˜200 nm) of agarose (0.66 M CaCl₂, 8.8 mMHEPES, Ph 7.0) is spin coated (3000× rpm, 30 s) on the coverslip and isincubated in 3 mM DPhPC in oil. The lipid coated droplet, upon contactwith the agarose, spontaneously forms a single DHB. The ground electrode(Ag/AgCl), which forms a closed circuit with the electrode (Ag/AgCl) inthe agarose, is stabbed into the droplet. The voltage protocols areapplied with a patch clamp amplifier (Axopatch 200B, Molecular Devices).αHL inside the droplet spontaneously inserts into DHB and the iontransport is detected both electrically (FIG. 6) and optically (FIG. 1d).

HHBa Measurements. Upon finishing loading, the chip is flipped andplaced on the electrode (FIG. 3b , FIG. 21) and the HHBa formsspontaneously when the chip is annealed with the substrate agarose in 3mM lipid/oil. The voltage protocols are applied with a patch clampamplifier (Axopatch 200B, Molecular Devices). Nanopore activities in theHHBa can be monitored with TIRF microscopy. 30 ms exposure time isnormally used to compensate the lower signal/noise ratio in a HHBameasurement. Due to the limited field of view, only 4 compartments in aHHBa chip can be recorded simultaneously.

Supplementary Materials

The lipid/oil used in this paper is defined as1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti PolarLipids) dissolved in oil.

Oil: 1:1 (v:v) mixture of hexadecane (Sigma-Aldrich) and silicone oilAR20 (Sigma-Aldrich).

To dissolve lipid in oil, the DPhPC powder is first dissolved in pentane(Sigma-Aldrich) in a 7 mL glass vial. It is then air dried with nitrogengas to form a thin film of lipid on the inner wall of the vial. Thelipid film is desiccator treated for more than 4 hours to remove theresidual pentane. Finally the lipid film is dissolved in the oil.Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich), agarose forroutine use (Sigma-Aldrich), agarose low melting point (Sigma-Aldrich),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)(Sigma-Aldrich), Potassium Chloride (Sigma-Aldrich), Calcium Chloride(Sigma-Aldrich), Pentane (Sigma-Aldrich), SU-8 2035 photoresist(MicroChem),Poly-methylmethacrylate (PMMA 495 a5) (MicroChem),Poly-dimethylsiloxane (PDMS, Sylgard 184) (Dow Corning), Fluo-8 (ABDBioquest), Chelex (BioRadchelex 100 Resin, Biotechnology Grade, 100-200mesh), Streptavidine (New England Biolabs) were used as received withoutfurther purification.

DNA (ATDbio) and RNA (IDTDNA) samples were purchased with HPLCpurification service and used without further purificaiton.

The protein nanopores used in this paper are αHL WT. The αHL heptamerprotein is E. Coli expressed and purified based on the publishedprotocols¹.

Supplementary Methods

1 | Buffer Preparation

Both the potassium chloride buffer (1.5 M KCl, 10 mM HEPES, PH 7.0) andthe calcium chloride buffer (0.75 M CaCl2, 10 mM HEPES, PH 7.0) wereprepared and membrane filtered (0.2 μm cellulose acetate, Nalgene) priorto use. The potassium chloride buffer used in any fluorescencemeasurement needs to be treated with Chelex 100 resin for overnight tominimize the divalent cation contaminations.

2 | DNA Sample Preparation

DNA samples are dissolved in DNase/RNase free water prior to use.Streptavidine and biotinlated ssDNA (C40, X3 or X5) are mixed with 1:1molar ratio in the potassium chloride buffer (1.5 M KCl, 10 mM EDTA, 10mM HEPES, PH 7.0, chelex treated) and incubated at 4° C. for 20 min toform the biotin/streptavidine tethering.

3 | miRNA/Probe Annealing

miRNA samples are dissolved in DNase/RNase free water prior to use. ThemiRNA and the probe are mixed with 1:1 molar ratio in the potassiumchloride buffer (1.5 M KCl, 10 mM EDTA, 10 mM HEPES, PH 7.0, chelextreated). To form probe/miRNA hybridization, the mixed solution isheated to 95° C. for 5 min and gradually cooled down from 65° C. to 25°C. with a rate of −5° C/min in a thermal cycler (Veriti, LifeTechnologies).

4 | TIRF Microscopy

TIRF measurements are performed with a Nikon Eclipse Ti microscopeequipped with a 60× oil immersion objective (Plan Apo TIRF, Nikon). Thefluorescence is excited by a 473 nm Argon ion laser (Shanghai DreamLaser Technologies) and imaged with an electron-multiplying CCD camera(Ixon3, Andor). In the TIRF recording, the full field of view is 150 μmby 150 μm. Parameters like the exposure time, EM gain and the binningsize are optimized to achieve the best S/N ratio in specific recordings.The highest recording rate that has been tested is 3 ms/frame.

5 | Fluorescent Trace Extraction and Normalization

The fluorescent images are recorded in .sif format (Andor Solis) andanalyzed by a home-made labview program. The program performs dataanalysis numerically by analyzing the raw image files as pixel values ina data array. The fluorescent trace can be extracted by either adding upthe pixel values of the fluorescent spots or by 2d Gaussian fitting. Dueto the long computation time of the fitting, all the fluorescent tracesin this paper are extracted by adding up the pixel values. 2d Gaussianfitting is performed only for demonstration (FIG. 9) and tracking (FIG.10). To minimize fluctuations from the laser illumination, thefluorescent intensity is normalized by the background fluorescencearound the pore (a donut shaped local background area) and this will getrid of most low frequency fluctuations in the fluorescent trace. Theabsolute value of OPC readout has huge measurement variations due to thedifferent optics conditions from time to time. However, by normalizingthe fluorescent trace with reference levels, such as the open porefluorescence at −50 mV and 0 mV (Fref0 and Fref1), this variation issignificantly minimized. This normalization process follows the formulabelow.

$F_{norm} = \frac{F_{raw} - F_{{ref}\; 0}}{F_{{ref}\; 1} - F_{{ref}\; 0}}$

6 | Planar Bilayer Measurements

Planar Lipid Membrane measurement is performed similar to the methodpublished before2. Briefly, lipid (DPhPC) bilayer forms across a Teflon(Good Fellow, 25 μm thick) aperture, which separates the Cis(electrically grounded) and the Trans chamber of the measurementapparatus (1 mL volume on both sides). Ionic current through a singleαHL in the PLM is patch-clamp recorded (Axopatch 200B, MolecularDevices) with a sampling rate of 5 kHz (Digidata 1440A digitizer,Molecular Devices) and is low-pass filtered at 1 kHz. Streptavidinetethered ssDNA (267 nM) is added to the Cis chamber and the chamber ismagnetically stirred to achieve homogeneous sample distribution. Voltageprotocols (100 mV, 0.9 sec; −140 mV, 0.05 sec; 0 mV, 0.05 sec) arerepeated 1000 times to accumulate enough events for statistics. To mimicthe optical recording measurement, the PLM is recorded with asymmetricbuffer condition (Cis: 1.32 M KCl, 8.8 mM HEPES, Ph 7.0; Trans: 0.66 MCaCl2, 8.8 mM HEPES, Ph: 7.0).

7 | Photolithography

The photomask is designed (AutoCAD) and printed on a transparent film(JDphoto). Micropatterns of SU-8 pillars are fabricated according to thestandard photolithography protocols (MicroChem):

1. Spin coating: 1 mL of SU-8 2035 photoresist is spin coated on the6-inch silicon wafer with the speed of 500 rpm for 15 seconds followedwith 2000 rpm for 35 seconds.

2. Pre-bake: The wafer is baked at 60° C. for 2.5 minutes and 95° C. for7 minutes

3. Exposure: The wafer, which is covered with the photomask, is UVexposed (200 mJ/cm2) for 30 seconds.

4. Develop: The wafer is then sprayed and washed with the developer for6 minutes.

5. Wash: The developed wafer is cleaned with isopropanol and air driedwith nitrogen streams.

6. Hard Bake: The wafer is baked at 150° C. for 10 min to finalize thelithography process.

The thickness of the fabricated pillar structures is around 40 μmaccording to the protocol (MicroChem) and the micropatterned wafer canbe re-used for multiple times in the following soft lithography process.

8 | Soft Lithography

The soft lithography process is performed according to the publishedprotocols³. Briefly, PDMS base and the curing agent are mixed with 10:1volume ratio. The mixture is poured over the micropattered silicon wafermould in a petri-dish and degassed for 1 hour. The PDMS mixture with themould is then incubated in the 80° C. oven for 4 hours to get fullycasted. The casted PDMS elastomer can be peeled off from the wafer. ThisPDMS mould can be re-used for multiple times in the following hydrogelchip fabrication process (FIG. 20).

Supplementary Discussions

1 | Pore Densities Estimation (Theoretical Limit)

Assuming that the pores are assembled into an ideal hexagonal array,each hexagonal unit cell contains 1+3×(⅙)=3 nanopores. If the pore topore distance is d, then the area of each unit cell is:

$\begin{matrix}{{Area} = {\frac{3\sqrt{3}}{2}d^{2}}} & (1)\end{matrix}$

The area per pore is:

$\begin{matrix}{{{Area}\text{/}{pore}} = {\frac{\sqrt{3}}{2}d^{2}}} & (2)\end{matrix}$

According to the half height width of each pore (FIG. 11), the minimumpore to pore separation for independent optical recording is d=3 μm.

The estimated pore density is:

$\begin{matrix}{N = {\frac{\left( 10^{- 3} \right)^{2}}{\frac{\sqrt{3}}{2}\left( {3 \times 10^{- 6}} \right)^{2}} = {1.3 \times 10^{5}\mspace{25mu} {{pores} \cdot {mm}^{- 2}}}}} & (3)\end{matrix}$

2 | Pore Densities Estimation

The bilayer analysis in this study is performed with a larger pore topore separation ˜10 μm (FIG. 1d ).

The recording density is estimated as:

N=1.16×10⁴ pores·mm⁻²   (4)

3 | Sequencing Densities

Based on published results^(4, 5) the nanopore sequencing speed couldachieve up to 40 nucleotides per second. With a hexagonal nanopore arrayof 3 μm pore to pore separation, one can simply estimate the sequencingdata production rate as below

40×1.3×10⁵=5.2×10⁶nucleotides·mm⁻²   (5)

In principle, for the size of a human genome (˜3×10⁹ nucleotides), thesequencing can be finished in ˜15 min (900 s) within a ˜mm² sized array.

4 | Unzipping Kinetics Modelling

The miRNA unzipping kinetics is modelled similar as reported before^(6, 7). To minimize the parameters, the modelling of the unzippingprocess is simplified as a single step, first-order reaction as belowand fits to the experiment results well (FIG. 2e ). The rate constantfrom state A (hybridized state) to state B (unzipped state) is definedto be k.

$\begin{matrix}{A\overset{k}{\rightarrow}B} & (6)\end{matrix}$

In a macroscopic reaction,

$\begin{matrix}{\frac{d\lbrack A\rbrack}{dt} = {- {k\lbrack A\rbrack}}} & (7) \\{\frac{d\lbrack B\rbrack}{dt} = {k\lbrack A\rbrack}} & (8)\end{matrix}$

The solutions to the above equations are:

$\begin{matrix}{\lbrack A\rbrack = {\left\lbrack A_{0} \right\rbrack e^{- {kt}}}} & (9) \\{\lbrack B\rbrack = {\left\lbrack A_{0} \right\rbrack \left( {1 - e^{- {kt}}} \right)}} & (10) \\{\frac{d\lbrack B\rbrack}{dt} = {\left\lbrack A_{0} \right\rbrack {ke}^{- {kt}}}} & (11)\end{matrix}$

In single molecule kinetics, the probability density function (p.d.f.)for a single molecule which has changed from state A to B between thetime interval is t→t+Δt:

$\begin{matrix}{{p.d.{f(t)}} = {\frac{d\lbrack B\rbrack}{\left\lbrack A_{0} \right\rbrack {dt}} = {ke}^{- {kt}}}} & (12)\end{matrix}$

The rate constant k, which reflects the hybridization strength betweenthe miRNA and the probe, is a function of the temperature (T) and theapplied potential (V):

$\begin{matrix}{{k\left( {T,V} \right)} = {k_{0}\exp \mspace{11mu} \left( {- \frac{E_{a} - {q_{eff}V}}{k_{B}T}} \right)}} & (13)\end{matrix}$

Here k_(B), is the Boltzmann constant (8.62×10⁻⁵ eV/K).

We assume that the unzipping process is driven by a constant electricalforce. And the effective charge (q_(eff)) maintains constant during thewhole unzipping process. Under the applied potential, the effectiveactivation energy (E_(a)) is lowered by q_(eff)V. By rearrangingequation (13):

$\begin{matrix}{{\ln (k)} = {{\frac{q_{eff}}{k_{B}T}\; V} - \frac{E_{a}}{k_{B}T} + {\ln \left( k_{0} \right)}}} & (14)\end{matrix}$

It is obvious that and V have a linear relationship and the fitted slopeequals to

$\frac{q_{eff}}{k_{B}T}.$

From the voltage dependence results, the fitted slope (a) equals to 22.0In (s⁻¹) Assuming that T=300K:

q_(eff)=ak_(B)T=0.57e   (15)

The qualitative model suggested here is too simple for accurateestimation of the effective charge. It is also believed that theeffective charge is estimated lower due to the different environmentinside a nanopore than in the buffer⁶.

TABLE 1 The nucleic acid abbreviations and sequences. ″X″in the sequence represents the abasic DNA nucleotide. Abbre- viationsNucleic Acid Sequence X

3′-Biotin-TEG-CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCXXXXXCCCCCCCCCCC-5′ X

3′-Biotin-TEG-CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCXXXCCCCCCCCCCCC-5′ C

3′-Biotin-TEG-CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-5′Plet7a 3′-C

-ACTCCATCATCCAACATATCAA-C

-5′ Plet7i 3′-C

-ACTCCATCATCAAACACGACAA-C

-5′ Let7a 3′-UUGAUAUGUUGGAUGAUGGAGU-5′ Let7i3′-UUGUCGUGUUUGAUGAUGGAGU-5′

indicates data missing or illegible when filed

TABLE 2 The statistics of the residual fluorescence by DNA blockages.The fluorescent amplitude is normalized according to the two referencelevels (open pore fluorescence at 0 mV and −50 mV). Mean FluorescentStandard Intensity/a.u. Deviation/a.u. Counts C₄₀ 0.38684 0.08433 132 X₃0.56737 0.06964 140 X₅ 0.68791 0.07229 121

TABLE 3 The statistics of the residual current by DNA blockages. Themean residual current and standard deviation is calculated from theGaussian fitting of the histogram (FIG. 14). Mean Residual Current/pAStandard Deviation/pA C₄₀ 8.02 0.13 X₃ 9.49 0.11 X₅ 10.46 0.12

TABLE 4 The statistics of the mean duration time for a full miRNAunzipping cycle. The large standard deviation is resulted from theexponential distribution of the duration times. T1/ms σT1/ms T2/msσT2/ms T3/ms σT3/ms Counts Plet7i/Let7i 393.41 769.20 957.40 1287.51125.63 284.87 541 Plet7i/Let7a 39.13 134.21 306.14 506.16 49.25 73.55350 Plet7a/Let7a 315.98 695.61 521.73 781.33 84.2 230.37 378Plet7a/Let7i 43.68 109.59 476.17 660.77 90.86 273.04 561 Let7i 0 0832.15 1263.18 128.37 177.75 87 Let7a 0 0 707.22 786.37 47.67 113.23 59

TABLE 5 The rate constants for different Probe/miRNA combinations. Therate constant is calculated from the exponential fitting of thehistogram for T1 (FIG. 2e). Sequence complimentary hybridizationsbetween probe and miRNA generate low rate constant values while theunmatched counterparts generate high rate constant values. Plet7a/Let 7aPlet7i/Let 7a Plet7i/Let 7i Plet7a/Let 7i Rate 13.66 68.80 7.06 47.45Constant/s⁻¹

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1. A method for detection of analyte interaction with a channel moleculeheld in a membrane, comprising the optical detection of a modificationin the flux of a signal molecule as it passes through the channelmolecule by the action of a membrane potential, wherein the modificationin the flux is caused by at least partial blockage of the channelmolecule by the analyte.
 2. The method according to claim 1, whereinmultiple channel molecules are provided in the membrane, and the methodcomprises the detection of analyte interaction with the multiple channelmolecules.
 3. The method according to claim 1 or claim 2, wherein themethod comprises the detection of analyte interaction with multiplechannel molecules held in multiple membranes, for example an array ofmembranes.
 4. The method according to any preceding claim, wherein anoptical field of the optical detection means is capable of encompassingand detecting optical signals/emissions across an array of membranes andchannel molecules.
 5. The method according to any preceding claim,wherein the membrane potential is provided by a single means.
 6. Themethod according to any preceding claim, wherein the membrane comprisesa bilayer of amphipathic molecules.
 7. The method according to anypreceding claim, wherein the channel molecule(s) comprises or consistsof nanopores.
 8. The method according to any preceding claim, whereinthe channel molecule comprises a biological molecule.
 9. The methodaccording to any preceding claim, wherein the channel molecule comprisesalpha-hemolysin (α-HL) or Mycobacterium smegmatis porin A (MspA); ormodified variants thereof.
 10. The method according to any of claims 1to 7, wherein the channel molecule(s) are synthetic nanopores.
 11. Themethod according to any preceding claim, wherein the signal molecule isa first signal-associated molecule provided on one side of the membrane,wherein the first signal-associated molecule is capable of flux throughthe channel molecule by the action of the membrane potential across themembrane; and a second signal-associated molecule is provided on theopposing side of the membrane relative to the first signal-associatedmolecule, wherein the first and/or second signal associated moleculesare arranged to emit an optical signal when in contact.
 12. The methodaccording to any preceding claim, wherein the signal molecule isarranged to interact with one or more other signal associated moleculesto cause or provide optical emission for optical detection after passingthrough the channel molecule.
 13. The method according to any precedingclaim, wherein the signal molecule comprises or consists of an ion, suchas Ca²⁺.
 14. The method according to claim 12 or 13, wherein the secondsignal-associated molecule comprises a calcium indicator.
 15. The methodaccording to any of claims 12 to 14, wherein the secondsignal-associated molecule comprises any molecule selected from thegroup comprising fura-2; indo-1; fluo-3; fluo-4; Calcium Green-1;Fluo-8; and Fluo-4; or combinations thereof.
 16. The method according toany preceding claim, wherein the analyte interaction with a channelmolecule comprises any of the interactions selected from the groupcomprising analyte flux through the channel molecule; analyte insertioninto the channel molecule; analyte blocking of the channel; analytemediated change in conformation of the channel molecule; stochasticblocking of the channel molecule by the analyte; and specific blockingof the channel molecule by the analyte; or combinations thereof.
 17. Themethod according to any preceding claim, wherein the analyte comprisesor consists of a biological molecule.
 18. The method according to anypreceding claim, wherein the analyte comprises or consists of a peptide,protein, or polynucleotide.
 19. The method according to any precedingclaim, wherein the method is for optical screening of a polynucleotidepresent in one or more samples, wherein the analyte is a polynucleotide.20. The method according to claim 19, wherein the polynucleotide analytecomprises a template polynucleotide arranged to hybridise with a targetpolynucleotide in the sample(s).
 21. The method according to claim 20,wherein a reduction in the flux of the signal molecule is indicative ofa successful hybridisation with the template polynucleotide as thehybridised double stranded polynucleotide at least partially blocks thechannel molecule.
 22. The method according to any of claims 19 to 21,wherein the method of optical screening comprises parallel screening ofmultiple samples and/or comprises the use of multiple templatepolynucleotides.
 23. The method according to any of claims 19 to 22,wherein the method for optical screening is carried out in an array ofmembranes.
 24. The method according to any of claims 1 to 18, whereinthe method is for optical polymer sequencing.
 25. The method accordingto claim 24, wherein the method is for optical polynucleotidesequencing.
 26. The method according to claim 24 or claim 25, whereinthe optical sequencing is parallel sequencing in an array.
 27. Themethod according to claim 25 or claim 26, wherein the modification inthe flux is caused by blockage of the channel molecule by thepolynucleotide bases as they pass through the channel molecule, whereindifferent bases are distinguishable by different levels of flux blockageor interference, which can be correlated to the individual bases.
 28. Abilayer array comprising: opposing hydrogel surfaces provided by a firstcomponent comprising an array of discrete hydrogel surfaces opposing ahydrogel surface of a second component; wherein the opposing hydrogelsurfaces are immersed in a hydrophobic medium comprising amphipathicmolecules, and wherein the opposing hydrogel surfaces are spaced apartsufficiently to provide bilayers of the amphipathic molecules formedtherebetween; channel molecules in the bilayers; a means arranged toprovide a membrane potential across the bilayers.
 29. The bilayer arrayaccording to claim 28, wherein the array of hydrogel surfaces isprovided on an array of pillars extending from the first component. 30.The bilayer array according to claim 29, wherein the pillars compriseand/or are formed of a hydrogel.
 31. The bilayer array according to anyof claims 28 to 30, wherein a discrete hydrogel surface of the firstcomponent is less than about 200 microns in diameter.
 32. The bilayerarray according to claim 31, wherein a discrete hydrogel surface of thefirst component is about 100 microns in diameter, or less.
 33. Thebilayer array according to any of claims 28 to 32, wherein the totalbilayer area provided by the array of bilayers is at least 1 mm². 34.The bilayer array according to any of claims 28 to 33, wherein the firstcomponent comprising an array of hydrogel surfaces further comprises abarrier material arranged to prevent cross-contamination of reagentsand/or channel molecules between adjacent hydrogel surfaces.
 35. Thebilayer array according to claim 34, wherein the barrier material isarranged between the hydrogel surfaces, for example between the pillars.36. The bilayer array according to any of claims 28 to 35, wherein thefirst component is supported by a plate.
 37. The bilayer array accordingto any of claims 28 to 36, wherein the second component compriseshydrogel, such as hydrogel layer.
 38. The bilayer array according to anyof claims 28 to 37, wherein the second component comprises a plate. 39.The bilayer array according to any of claims 28 to 38, wherein the meansfor providing a membrane potential comprises cathode and anodeelectrodes.
 40. The bilayer array according to any of claims 28 to 39,wherein the bilayer array comprises only a single set of anodes andcathodes for providing membrane potential across all the bilayers in thearray.
 41. The bilayer array according to any of claims 28 to 40,wherein the hydrogel comprise or consist of less than 5% (w/v) agarose.42. The bilayer array according to any of claims 28 to 41, wherein thefirst component hydrogel comprises a potassium chloride buffer and thesecond component hydrogel comprises a calcium chloride buffer; or viceversa.
 43. The bilayer array according to claim 42, wherein the bufferfurther comprises EDTA.
 44. A method of manufacturing a bilayer arraycomprising providing a mould for casting a hydrogel first component,wherein the mould comprises an array of cavities for forming thehydrogel first component with an array of hydrogel pillars; pouring aliquid arranged to set into a hydrogel into the mould; laying a plateonto the surface of the liquid and setting the liquid to form thehydrogel first component; removing the mould from the hydrogel firstcomponent; optionally coating a barrier material on the hydrogel firstcomponent, between the hydrogel pillars; adding a reagents and/orsamples to one or more hydrogel pillars; providing a second componentcomprising a hydrogel surface on a plate; submerging the hydrogelsurfaces of the first and second components in a volume of hydrophobicmedium comprising amphipathic molecules, thereby forming a monolayer ofamphipathic molecules on the hydrogel surfaces of the first and secondcomponents; bringing the first and second components into an opposingposition relative to each other such that the monolayers of amphipathicmolecules form a bilayer.
 45. A bilayer array formed by the methodaccording to claim
 44. 46. A method of manufacturing a component for abilayer array comprising: providing a mould for casting a hydrogelcomponent, wherein the mould comprises an array of cavities for castinga hydrogel component comprising an array of pillars; pouring a liquidarranged to set into a hydrogel into the mould; laying a plate onto thesurface of the liquid and setting the liquid to form a hydrogelcomponent; removing the mould from the hydrogel component; andoptionally coating a barrier material onto the hydrogel component,between the pillars.
 47. A component for a bilayer array formed by themethod according to claim
 46. 48. A hydrogel component for a bilayerarray comprising an array of pillars extending from the surface of thehydrogel component, and a barrier material coating the surface of thecomponent between the pillars.
 49. A kit for forming a bilayer arraycomprising: a first component comprising an array of discrete hydrogelsurfaces; and a second component comprising a hydrogel layer.
 50. A kitfor manufacturing a bilayer array comprising: a first mould for castinga first component comprising an array of discrete hydrogel surfaces; anda second mould for casting a second component comprising a hydrogellayer.
 51. A method of optical screening of a polynucleotide present inone or more samples, the method comprising the steps: providing amembrane comprising channel molecules held in the membrane; providingone or more template polynucleotides and one or more polynucleotidesfrom one or more samples on one side of the membrane; providing amembrane potential across the membrane, such that it provides flux of asignal molecule through the channel molecules; detecting hybridisation,or lack thereof, of the one or more template polynucleotides withpolynucleotide in the one or more samples by optically detecting anymodification in flux of the signal molecule through the channelmolecules.
 52. The method of optical screening according to claim 51,comprising the simultaneous or parallel screening of multiple samplesand/or comprising the use of multiple template polynucleotides.
 53. Useof the bilayer array according to any of claims 28 to 43, for opticalpolymer sequencing or analyte analysis.
 54. The use according to claim53, wherein the optical polymer sequencing or analyte analysis isparallel sequencing or analysis in an array.
 55. A method for detectionof analyte interaction with a nanopore held in a bilayer of amphipathicmolecules, comprising: providing a bilayer of amphipathic molecules,wherein the bilayer comprises one or more nanopores; providing a firstsignal-associated molecule on one side of the bilayer, wherein the firstsignal-associated molecule is capable of flux through the nanopore(s) bythe action of a membrane potential across the bilayer; providing asecond signal-associated molecule on the opposing side of the bilayerrelative to the first signal-associated molecule, wherein the firstand/or second signal associated molecules are arranged to emit anoptical signal when in contact; providing an analyte on at least oneside of the bilayer; applying a membrane potential across the bilayersuch that the first signal-associated molecule is transported throughthe nanopore and interacts with the second signal-associated molecule toemit an optical signal; detecting the optical signal; and detecting anymodification, or lack thereof, in the optical signal as the flux of thefirst signal-associated molecule is modified by at least partialblocking of the nanopore by the analyte as it interacts with thenanopore.
 56. A method for detection of analyte interaction with achannel molecule held in a membrane, a bilayer array, the use, or amethod of manufacture, substantially as described herein, with referenceto the accompanying drawings.